Phenotypic analysis of astrocytes derived from glial restricted precursors and their impact on axon regeneration.

Although astrocytes are involved in the production of an inhibitory glial scar following injury, they are also capable of providing neuroprotection and supporting axonal growth. There is growing appreciation for a diverse and dynamic population of astrocytes, specified by a variety of glial precursors, whose function is regulated regionally and temporally. Consequently, the therapeutic application of glial precursors and astrocytes by effective transplantation protocols requires a better understanding of their phenotypic and functional properties and effective protocols for their preparation. We present a systematic analysis of astrocyte differentiation using multiple preparations of glial-restricted precursors (GRP), evaluating their morphological and phenotypic properties following treatment with fetal bovine serum (FBS), bone morphogenetic protein 4 (BMP-4), or ciliary neurotrophic factor (CNTF) in comparison to controls treated with basic fibroblast growth factor (bFGF), which maintains undifferentiated GRP. We found that treatments with FBS or BMP-4 generated similar profiles of highly differentiated astrocytes that were A2B5-/GFAP+. Treatment with FBS generated the most mature astrocytes, with a distinct and near-homogeneous morphology of fibroblast-like flat cells, whereas BMP-4 derived astrocytes had a stellate, but heterogeneous morphology. Treatment with CNTF induced differentiation of GRP to an intermediate state of GFAP+cells that maintained immature markers and had relatively long processes. Furthermore, astrocytes generated by BMP-4 or CNTF showed considerable experimental plasticity, and their morphology and phenotypes could be reversed with complementary treatments along a wide range of mature-immature states. Importantly, when GRP or GRP treated with BMP-4 or CNTF were transplanted acutely into a dorsal column lesion of the spinal cord, cells from all 3 groups survived and generated permissive astrocytes that supported axon growth and regeneration of host sensory axons into, but not out of the lesion. Our study underscores the dynamic nature of astrocytes prepared from GRP and their permissive properties, and suggest that future therapeutic applications in restoring connectivity following CNS injury are likely to require a combination of treatments.

Acute administration of AMPA/Kainate blocker combined with delayed transplantation of neural precursors improves lower urinary tract function in spinal injured rats.

To evaluate bladder function recovery after spinal cord injury (SCI) in response to a combination treatment of an acutely administered AMPA/kainate receptor antagonist and delayed transplantation of neuronal precursors. Female rats received a contusion injury at T8/9. The AMPA/kainate receptor antagonist NBQX was directly administered into the lesion site immediately after injury. Nine days post-injury, NRP/GRP were delivered into the lesion site. Controls received NRP/GRP grafts only or no treatment (OP-Controls). Animals underwent bladder function testing during the course of the experiment and at the endpoint. Motor function was evaluated as well. After sacrifice, histological analysis of lesion site and lumbosacral spinal cord regions was performed. Rats receiving the combined treatment (NBQX&NRP/GRP) had voided volumes/micturition resembling that of normal animals and showed greater improvement of urodynamic parameters, compared to NRP/GRP alone or OP-Controls. Similarly, NBQX&NRP/GRP induced more spouting, regeneration or sparing of descending projections to the lumbosacral cord. The density of primary afferent projections at the lumbosacral spinal cord in rats with combined treatments was similar to that of NRP/GRP alone with decreased sprouting of primary afferents in lumbosacral cord, compared to OP-Control. Immunohistochemical evaluation revealed that the combined treatment reduced the size of the lesion to a greater extent than NRP/GRP alone or OP-Controls. NRP/GRP with and without NBQX produced a significant recovery of hindlimb compared to OP-Controls. In conclusion, transplants of NRP/GRP combined with NBQX promote recovery of micturition function following spinal cord injury, likely through increased neuroprotection.

A pilot study of poly(N-isopropylacrylamide)-g-polyethylene glycol and poly(N-isopropylacrylamide)-g-methylcellulose branched copolymers as injectable scaffolds for local delivery of neurotrophins and cellular transplants into the injured spinal cord.

OBJECT: The authors investigated the feasibility of using injectable hydrogels, based on poly(N-isopropylacrylamide) (PNIPAAm), lightly cross-linked with polyethylene glycol (PEG) or methylcellulose (MC), to serve as injectable scaffolds for local delivery of neurotrophins and cellular transplants into the injured spinal cord. The primary aims of this work were to assess the biocompatibility of the scaffolds by evaluating graft cell survival and the host tissue immune response. The scaffolds were also evaluated for their ability to promote axonal growth through the action of released brain-derived neurotrophic factor (BDNF). METHODS: The in vivo performance of PNIPAAm-g-PEG and PNIPAAm-g-MC was evaluated using a rodent model of spinal cord injury (SCI). The hydrogels were injected as viscous liquids into the injury site and formed space-filling hydrogels. The host immune response and biocompatibility of the scaffolds were evaluated at 2 weeks by histological and fluorescent immunohistochemical analysis. Commercially available matrices were used as a control and examined for comparison. RESULTS: Experiments showed that the scaffolds did not contribute to an injury-related inflammatory response. PNIPAAm-g-PEG was also shown to be an effective vehicle for delivery of cellular transplants and supported graft survival. Additionally, PNIPAAm-g-PEG and PNIPAAm-g-MC are permissive to axonal growth and can serve as injectable scaffolds for local delivery of BDNF. CONCLUSIONS: Based on the results, the authors suggest that these copolymers are feasible injectable scaffolds for cell grafting into the injured spinal cord and for delivery of therapeutic factors.

Transplantation of human glial restricted progenitors and derived astrocytes into a contusion model of spinal cord injury.

Transplantation of neural progenitors remains a promising therapeutic approach to spinal cord injury (SCI), but the anatomical and functional evaluation of their effects is complex, particularly when using human cells. We investigated the outcome of transplanting human glial-restricted progenitors (hGRP) and astrocytes derived from hGRP (hGDA) in spinal cord contusion with respect to cell fate and host response using athymic rats to circumvent xenograft immune issues. Nine days after injury hGRP, hGDA, or medium were injected into the lesion center and rostral and caudal to the lesion, followed by behavioral testing for 8 weeks. Both hGRP and hGDA showed robust graft survival and extensive migration. The total number of cells increased 3.5-fold for hGRP, and twofold for hGDA, indicating graft expansion, but few proliferating cells remained by 8 weeks. Grafted cells differentiated into glia, predominantly astrocytes, and few remained at progenitor state. About 80% of grafted cells around the injury were glial fibrillary acidic protein (GFAP)-positive, gradually decreasing to 40-50% at a distance of 6 mm. Conversely, there were few graft-derived oligodendrocytes at the lesion, but their numbers increased away from the injury to 30-40%. Both cell grafts reduced cyst and scar formation at the injury site compared to controls. Microglia/macrophages were present at and around the lesion area, and axons grew along the spared tissue with no differences among groups. There were no significant improvements in motor function recovery as measured by the Basso, Beattie, and Bresnahan (BBB) scale and grid tests in all experimental groups. Cystometry revealed that hGRP grafts attenuated hyperactive bladder reflexes. Importantly, there was no increased sensory or tactile sensitivity associated with pain, and the hGDA group showed sensory function returning to normal. Although the improved lesion environment was not sufficient for robust functional recovery, the permissive properties and lack of sensory hypersensitivity indicate that human GRP and astrocytes remain promising candidates for therapy after SCI.

Induction of filopodia-like protrusions by transmembrane agrin: role of agrin glycosaminoglycan chains and Rho-family GTPases.

Filopodia sense the extracellular environment and direct movement in many cell types, including neurons. Recent reports suggest that the transmembrane form of the widely expressed proteoglycan agrin (TM-agrin) regulates formation and stability of neuronal filopodia. In order to elucidate the mechanism by which TM-agrin regulates filopodia, we investigated the role of agrin's glycosaminoglycan (GAG) chains in the induction of filopodia formation by TM-agrin over-expression in hippocampal neurons, and in the induction of filopodia-like processes in COS7 cells. Deletion of the GAG chains of TM-agrin sharply reduced formation of filopodia-like branched retraction fibers (BRFs) in COS7 cells, with deletion of the heparan sulfate GAG chains being most effective, and eliminated filopodia induction in hippocampal neurons. GAG chain deletion also reduced the activation of Cdc42 and Rac1 resulting from TM-agrin over-expression. Moreover, dominant-negative Cdc42 and Rac1 inhibited BRF formation. Lastly, over-expression of TM-agrin increased the adhesiveness of COS7 cells and this increase was reduced by deletion of the GAG chains. Our results suggest that TM-agrin regulates actin-based protrusions in large part through interaction of its GAG chains with extracellular or transmembrane proteins, leading to the activation of Cdc42 and Rac1.

Secretion profile of human bone marrow stromal cells: donor variability and response to inflammatory stimuli.

Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli.

Neurite outgrowth of neural progenitors in presence of inhibitory proteoglycans.

Attempts to promote host regeneration after spinal cord injury (SCI) have often resulted in poor axon extension due to formation of a glial scar, which creates a dense physical barrier around the injury and contains molecules that inhibit regeneration and repair of adult injured axons. Previous studies have shown that, while transplants of multipotent neural stem cells (NSC) integrate poorly in the injury site, the use of neuronal-restricted precursor cells (NRP) together with glial-restricted precursor cells (GRP) allow differentiation and integration of neurons, possibly because NRP are able to overcome chondroitin sulfate proteoglycan (CSPG) inhibition. To investigate this possibility, we grew mixed cultures of NRP/GRP on CSPG at inhibitory concentrations, using embryonic hippocampal cultures as controls. We found that NRP/GRP grown on CSPG survive and differentiate into neurons with no significant changes in neurite length, relative to growth on control polylysine substrate, and in contrast to a significant inhibition of axon growth in hippocampal cultures grown on CSPG-coated substrate. There was, however, a significant decrease in neurite number and branching in both cultures, indicating that CSPG also has important effects on neuronal morphology. These data suggest that embryonic neurons supported by glial cells derived from NRP/GRP transplants are less sensitive to inhibitory effects of CSPG in the glial scar, and are thus an appropriate source for neuronal cell replacement and reconnection of damaged circuits after SCI.

Promoting directional axon growth from neural progenitors grafted into the injured spinal cord.

Spinal cord injury (SCI) is a devastating condition characterized by disruption of axonal connections, failure of axonal regeneration, and loss of motor and sensory function. The therapeutic promise of neural stem cells has been focused on cell replacement, but many obstacles remain in obtaining neuronal integration following transplantation into the injured CNS. This study investigated the neurotransmitter identity and axonal growth potential of neural progenitors following grafting into adult rats with a dorsal column lesion. We found that using a combination of neuronal and glial restricted progenitors (NRP and GRP) produced graft-derived glutamatergic and GABAergic neurons within the injury site, with minimal axonal extension. Administration of brain-derived neurotrophic factor (BDNF) with the graft promoted modest axonal growth from grafted cells. In contrast, injecting a lentiviral vector expressing BDNF rostral into the injured area generated a neurotrophin gradient and promoted directional growth of axons for up to 9 mm. Animals injected with BDNF lentivirus (at 2.5 and 5.0 mm) showed significantly more axons and significantly longer axons than control animals injected with GFP lentivirus. However, only the 5.0-mm-BDNF group showed a preference for extension in the rostral direction. We concluded that NRP/GRP grafts can be used to produce excitatory and inhibitory neurons, and neurotrophin gradients can guide axonal growth from graft-derived neurons toward putative targets. Together they can serve as a building block for neuronal cell replacement of local circuits and formation of neuronal relays.

Transplantation of human marrow stromal cells and mono-nuclear bone marrow cells into the injured spinal cord: a comparative study.

STUDY DESIGN: Two groups of 6 rats received dorsolateral funiculotomies followed by direct injection of bone marrow stromal cells (MSC) or mono-nuclear fraction of bone marrow (mnBM). Animals were killed at 4 or 21 days. OBJECTIVE: Cellular transplantation is a promising treatment strategy for spinal cord injury (SCI); however, most cells need to be cultured before transplantation introducing burdensome steps for clinical application. Cells immediately available for transplantation, like mnBM, would be preferable. SUMMARY OF BACKGROUND DATA: Previous studies have shown that MSC transplants promote protection and repair after SCI. MSC are attractive for transplantation because of easy isolation and availability of autologous sources. MSC are derived from whole bone marrow, purified and expanded in culture for a period of at least 2 weeks. Alternatively, mnBM could be used for transplantation. mnBM derived from bone marrow from through simple centrifugation can be reimplantated within hours; however, the presence of immune cells may be problematic. METHODS: Cultured MSC or mnBM from human donors were acutely transplanted into SCI. After sacrifice, spinal cord sections were histologically analyzed for presence of graft-derived immune cells, host immune response, tissue sparing, glial scar formation, and grafting efficacy. RESULTS: mnBM did not give rise to mature immune cells after transplantation into SCI, or evoke an increased host immune response or tissue loss compared to MSC-transplanted animals. In contrast, host macrophage/microglia response was increased early after MSC transplantation, perhaps due to exposure of cells to serum-containing media. The glial scar was less prominent after mnBM transplantation at day 4. At 21 days, differences had subsided and MSC and mnBM macrophage responses and effects on glial scarring were comparable. MSC and mnBM engraftment efficiencies were also similar. CONCLUSION: The use of mnBM is a viable alternative to MSC for transplantation into SCI and may dramatically ease clinical translation.

Grafting of human bone marrow stromal cells into spinal cord injury: a comparison of delivery methods.

STUDY DESIGN: Three groups of 6 rats received subtotal cervical spinal cord hemisections followed with marrow stromal cell (MSC) transplants by lumbar puncture (LP), intravenous delivery (IV), or direct injection into the injury (control). Animals survived for 4 or 21 days. OBJECTIVE: Cell therapy is a promising strategy for the treatment of spinal cord injury (SCI). The mode of cell delivery is crucial for the translation to the clinic. Injections directly into the parenchyma may further damage already compromised tissue; therefore, less invasive methods like LP or IV delivery are preferable. SUMMARY OF BACKGROUND DATA: Human MSC are multipotent mesenchymal adult stem cells that have a potential for autologous transplantation, obviating the need for immune suppression. Although previous studies have established that MSC can be delivered to the injured spinal cord by both LP and IV, the efficacy of cell delivery has not been directly compared with respect to efficacy of delivery and effects on the host. METHODS: Purified MSC from a human donor were transplanted into the CSF at the lumbar region (LP), into the femoral vein (IV), or directly into the injury (control). After sacrifice, spinal cord sections were analyzed for MSC graft size, tissue sparing, host immune response, and glial scar formation, using specific antibodies and Nissl-myelin staining. RESULTS: LP delivery of MSC to the injured spinal cord is superior to IV delivery. Cell engraftment and tissue sparing were significantly better after LP delivery, and host immune response after LP delivery was reduced compared with IV delivery. CONCLUSION: LP is an ideal minimally invasive technique to deliver cellular transplants to the injured spinal cord. It is superior to IV delivery and, together with the potential for autologous transplantation, lends itself for clinical application.

In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair.

Nervous tissue engineering in combination with other therapeutic strategies is an emerging trend for the treatment of different CNS disorders and injuries. We propose to use poly(N-isopropylacrylamide)-co-poly(ethylene glycol) (PNIPAAm-PEG) as a minimally invasive, injectable scaffold platform for the repair of spinal cord injury (SCI). The scaffold allows cell attachment, and provides mechanical support and a sustained release of neurotrophins. In order to use PNIPAAm-PEG as an injectable scaffold for treatment of SCI, it must maintain its mass and volume over time in physiological conditions. To provide mechanical support at the injury site, it is also critical that the engineered scaffold matches the compressive modulus of the native neuronal tissue. This study focused on studying the ability of the scaffold to release bioactive neurotrophins and matching the material properties to those of the native neuronal tissue. We found that the release of both BDNF and NT-3 was sustained for up to 4 weeks, with a minimal burst exhibited for both neurotrophins. The bioactivity of the released NT-3 and BDNF was confirmed after 4 weeks. In addition, our results show that the PNIPAAm-PEG scaffold can be designed to match the desired mechanical properties of the native neuronal tissue, with a compressive modulus in the 3-5 kPa range. The scaffold was also compatible with bone marrow stromal cells, allowing their survival and attachment for up to 31 days. These results indicate that PNIPAAm-PEG is a promising multifunctional scaffold for the treatment of SCI.

Stem cell delivery by lumbar puncture as a therapeutic alternative to direct injection into injured spinal cord.

OBJECT: Using cellular transplants to treat spinal cord injury is a promising therapeutic strategy, but transplants grafted directly into the injury site can further damage the already compromised cord. To avoid additional trauma and to simplify translation to the clinic, it is advantageous to use less invasive delivery methods. METHODS: The authors compared the efficacy of intrathecal cell delivery at the lumbar region (lumbar puncture [LP]) to direct injection into a thoracic contusion injury using a mixed population of lineage-restricted neural precursor cells. RESULTS: Direct injection resulted in a higher volume of neural precursor cells located throughout the injury site, whereas fewer LP-delivered cells accumulated at the dorsal aspect of the injured cord. Both grafting methods were neuroprotective, resulting in reduction of injury size and greater tissue sparing compared with controls. Functional recovery was evaluated by assessing motor and bladder function. Animals that received cells via direct injection performed significantly better in the open-field locomotor test than did operated controls, while LP-treated animals showed intermediate recovery of function that did not differ statistically from that of either operated controls or directly injected animals. Bladder function, however, was significantly improved in both directly injected and LP-treated animals. CONCLUSION: Grafting of stem cells via LP resulted in localized accumulation of cells at the injury site, neuroprotection, and modest recovery of function. Further optimization of the LP procedure by increasing the number of cells that are delivered and determining the optimal delivery schedule may further improve recovery to levels comparable to direct injection.

Effects of plating density and culture time on bone marrow stromal cell characteristics.

OBJECTIVE: Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. MATERIALS AND METHODS: Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations. RESULTS: We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. CONCLUSION: Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.

Combining motor training with transplantation of rat bone marrow stromal cells does not improve repair or recovery in rats with thoracic contusion injuries.

Previous studies have demonstrated that either transplantation of bone marrow stromal cells (MSC) or physical exercise regimens can elicit limited functional recovery following spinal cord injury, presumably through different mechanisms. The present study examined whether transplantation of MSC derived from transgenic Fischer alkaline phosphatase (AP) rats, in combination with exercise, would have synergistic effects leading to recovery of function that is greater than either alone. Adult female Sprague-Dawley rats received a moderate thoracic contusion injury and were divided into three groups: operated controls (Op-Control), MSC transplant recipients (MSC), and MSC transplant recipients plus exercise (MSC+Ex). Nine days after contusion, a Vitrogen matrix +/-one million MSC was injected into the lesion site in all animals. Immunosuppression with high doses of Cyclosporine A, required for MSC survival, was provided for all animals. Passive hindlimb exercise on motorized bicycles was applied 1 h/day, 3 days/week to the MSC+Ex group. A battery of behavioral tests was performed weekly to assess motor and sensory functions in all 3 groups for 12 weeks. Morphological evaluation included MSC survival, evidence of axonal growth into grafts, phenotypic analysis of MSC, and lesion/transplant size. The weight of the medial gastrocnemius muscle, a hindlimb muscle activated during stance, was used to identify extent of atrophy. No differences in motor recovery were found among the three groups. MSC survived 3 months after transplantation, indicating that the immunosuppression treatment was successful. The extent of survival was variable, and there was no correlation between MSC survival and behavioral scores. The matrix persisted, filling the lesion cavity, and some axons grew into the lesion/matrix but to a similar extent in all groups. There was no difference in lesion/matrix size among groups, indicating no neuroprotective effect on the host provided by the treatments. Immunocytochemical analysis provided no evidence that MSC differentiated into neurons, astrocytes or oligodendrocytes. Muscle mass of the medial gastrocnemius was diminished in the Op-Control group indicating significant atrophy, but was partially preserved in both the MSC and MSC+Ex groups. Our results indicate that combining the beneficial effects of rat MSC and this exercise protocol was not sufficient to enhance behavioral recovery.

Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord.

This study evaluates functional recovery after transplanting human bone marrow-derived stromal cells (BMSCs) into contusion models of spinal cord injury (SCI). The authors used a high-throughput process to expand BMSCs and characterized them by flow cytometry, ELISA, and gene expression. They found that BMSCs secrete neurotrophic factors and cytokines with therapeutic potential for cell survival and axon growth. In adult immune-suppressed rats, mild, moderate, or severe contusions were generated using the MASCIS impactor. One week following injury, 0.5 to 1 x 106 BMSCs were injected into the lesioned spinal cord; control animals received vehicle injection. Biweekly behavioral tests included the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB), exploratory rearing, grid walking, and thermal sensitivity. Animals receiving moderate contusions followed by BMSC grafts showed significant behavioral recovery in BBB and rearing tests when compared to controls. Animals receiving BMSC grafts after mild or severe contusion showed trends toward improved recovery. Immunocytochemistry identified numerous axons passing through the injury in animals with BMSC grafts but few in controls. BMSCS were detected at 2 weeks after transplantation; however, at 11 weeks very few grafted cells remained. The authors conclude that BMSCs show potential for repairing SCI. However, the use of carefully characterized BMSCs improved transplantation protocols ensuring BMSC, survival, and systematic motor and sensory behavioral testing to identify robust recovery is imperative for further improvement.

Lumbar puncture delivery of bone marrow stromal cells in spinal cord contusion: a novel method for minimally invasive cell transplantation.

Cell transplantation as a treatment for spinal cord injury is a promising therapeutic strategy whose effective clinical application would be facilitated by non-invasive delivery protocols. Cells derived from the bone marrow are particularly attractive because they can be obtained easily, expanded to large numbers and potentially used for autologous as well as allogeneic transplantation. In this study we tested the feasibility of a novel minimally invasive method--lumbar puncture (LP)--for transplanting bone marrow stromal stem cells (MSC) into a clinically relevant spinal cord contusion model. We further sought to determine optimal protocols for performing such minimally invasive cell transplantation. Sprague-Dawley rats received a moderate contusion injury at the midthoracic level followed by LP transplantation of MSC derived from transgenic rats that express the human placental alkaline phosphatase (AP) reporter gene. The recipients were analyzed histologically to evaluate the extent of cell delivery and survival at the injury site. We found that MSC delivered by LP reached the contused spinal cord tissues and exerted a significant beneficial effect by reducing cyst and injury size. Transplantation within 14 days of injury provided significantly greater grafting efficiency than more delayed delivery, and increasing MSC dosage improved cell engraftment. The techniques described here can easily be translated to patients, thus accelerating clinical application of stem cell therapies.

Axon growth and recovery of function supported by human bone marrow stromal cells in the injured spinal cord exhibit donor variations.

Bone marrow stromal cells (MSC) are non-hematopoietic support cells that can be easily derived from bone marrow aspirates. Human MSC are clinically attractive because they can be expanded to large numbers in culture and reintroduced into patients as autografts or allografts. We grafted human MSC derived from aspirates of four different donors into a subtotal cervical hemisection in adult female rats and found that cells integrated well into the injury site, with little migration away from the graft. Immunocytochemical analysis demonstrated robust axonal growth through the grafts of animals treated with MSC, suggesting that MSC support axonal growth after spinal cord injury (SCI). However, the amount of axon growth through the graft site varied considerably between groups of animals treated with different MSC lots, suggesting that efficacy may be donor-dependent. Similarly, a battery of behavioral tests showed partial recovery in some treatment groups but not others. Using ELISA, we found variations in secretion patterns of selected growth factors and cytokines between different MSC lots. In a dorsal root ganglion explant culture system, we tested efficacy of conditioned medium from three donors and found that average axon lengths increased for all groups compared to control. These results suggest that human MSC produce factors important for mediating axon outgrowth and recovery after SCI but that MSC lots from different donors vary considerably. To qualify MSC lots for future clinical application, such notable differences in donor or lot-lot efficacy highlight the need for establishing adequate characterization, including the development of relevant efficacy assays.

Analysis of allogeneic and syngeneic bone marrow stromal cell graft survival in the spinal cord.

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.

Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype.

Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron-like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [2002] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time-lapse studies. Similar neurite-like processes were induced by treating MSC with DMSO only or with actin filament-depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages.

Targeting of recombinant agrin to axonal growth cones.

Targeting of proteins to specific subcellular locations within pre- and postsynaptic neurons is essential for synapse formation. The heparan sulfate proteoglycan agrin orchestrates postsynaptic differentiation of the neuromuscular junction and may be involved in synaptic development and signaling in the central nervous system (CNS). Agrin is expressed as transmembrane and secretory isoforms with distinct N-termini. We examined the distribution of recombinant agrin in cultured motor and hippocampal neurons by transfection with agrin-GFP constructs. Immunostaining revealed a vesicular transport compartment within all neurites. Plasma membrane insertion and secretion of recombinant agrin were targeted to axonal growth cones of motor neurons; transmembrane agrin-GFP was targeted predominantly to axons and axonal growth cones in hippocampal neurons. We used agrin deletion mutants to show that axonal targeting of agrin depends on multiple domains that function in an additive fashion, including the very N-terminal portions and the C-terminal half of the molecule.